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Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

Hagemann S, Heil O, Lyko F, Brueckner B - PLoS ONE (2011)

Bottom Line: Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context.These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes.Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

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Related in: MedlinePlus

The majority of CGs resistant to drug-induced demethylation become demethylated in DKO cells.A, DKO cells show substantial differences to HCT116 cells in their methylation pattern; blue dots and numbers represent demethylated or hypermethylated CGs (DB≤−0.2 or ≥0.2). B, Boxplots show the demethylation (DB) in drug-treated HCT16 cells and DKO cells; black lines denote medians, notches the standard errors, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. C, Comparison of relative mean methylation of drug-treated cells and DKO cells as determined by global genomic methylation analysis (CE) and Infinium methylation analysis. D, Venn diagrams indicate overlapping demethylated CGs between drug-treated cells and DKO cells.
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pone-0017388-g003: The majority of CGs resistant to drug-induced demethylation become demethylated in DKO cells.A, DKO cells show substantial differences to HCT116 cells in their methylation pattern; blue dots and numbers represent demethylated or hypermethylated CGs (DB≤−0.2 or ≥0.2). B, Boxplots show the demethylation (DB) in drug-treated HCT16 cells and DKO cells; black lines denote medians, notches the standard errors, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. C, Comparison of relative mean methylation of drug-treated cells and DKO cells as determined by global genomic methylation analysis (CE) and Infinium methylation analysis. D, Venn diagrams indicate overlapping demethylated CGs between drug-treated cells and DKO cells.

Mentions: To further characterize the CGs resistant to drug-induced demethylation we obtained methylation profiles from HCT116 cells with strongly reduced levels of DNMT1 and complete loss of DNMT3B (DKO cells). Data analysis revealed pronounced demethylation in DKO cells with more than 85% of methylated CGs being demethylated (Figure 3A). DKO cells also showed the greatest degree of demethylation represented by median DB values of less than −0.55 relative to control cells (Figure 3B). In comparison, we observed significantly (P<2×10−16, Wilcoxon rank sum test) lower degrees of demethylation for AZA and DAC (Figure 3B).


Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

Hagemann S, Heil O, Lyko F, Brueckner B - PLoS ONE (2011)

The majority of CGs resistant to drug-induced demethylation become demethylated in DKO cells.A, DKO cells show substantial differences to HCT116 cells in their methylation pattern; blue dots and numbers represent demethylated or hypermethylated CGs (DB≤−0.2 or ≥0.2). B, Boxplots show the demethylation (DB) in drug-treated HCT16 cells and DKO cells; black lines denote medians, notches the standard errors, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. C, Comparison of relative mean methylation of drug-treated cells and DKO cells as determined by global genomic methylation analysis (CE) and Infinium methylation analysis. D, Venn diagrams indicate overlapping demethylated CGs between drug-treated cells and DKO cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049766&req=5

pone-0017388-g003: The majority of CGs resistant to drug-induced demethylation become demethylated in DKO cells.A, DKO cells show substantial differences to HCT116 cells in their methylation pattern; blue dots and numbers represent demethylated or hypermethylated CGs (DB≤−0.2 or ≥0.2). B, Boxplots show the demethylation (DB) in drug-treated HCT16 cells and DKO cells; black lines denote medians, notches the standard errors, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. C, Comparison of relative mean methylation of drug-treated cells and DKO cells as determined by global genomic methylation analysis (CE) and Infinium methylation analysis. D, Venn diagrams indicate overlapping demethylated CGs between drug-treated cells and DKO cells.
Mentions: To further characterize the CGs resistant to drug-induced demethylation we obtained methylation profiles from HCT116 cells with strongly reduced levels of DNMT1 and complete loss of DNMT3B (DKO cells). Data analysis revealed pronounced demethylation in DKO cells with more than 85% of methylated CGs being demethylated (Figure 3A). DKO cells also showed the greatest degree of demethylation represented by median DB values of less than −0.55 relative to control cells (Figure 3B). In comparison, we observed significantly (P<2×10−16, Wilcoxon rank sum test) lower degrees of demethylation for AZA and DAC (Figure 3B).

Bottom Line: Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context.These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes.Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

Show MeSH
Related in: MedlinePlus